STERILE FLUIDS

I. Introduction

Viruses which may be isolated from pleural, peritoneal and pericardial fluid are enteroviruses (Coxsackie Group A and B, poliovirus, and echoviruses). Viral isolation from joint fluid is exceptionally rare. Amniotic fluid will be set up for cytomegalovirus shell vial and tube culture. Specimens with requests for HSV or VZV also necessitate inoculation of shell vials. Refer to Appendix XV (Virus isolation and isolation and identification) to ensure the appropriate media are inoculated.

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II. Collection and Transport

Specimens should be collected in a clean, sterile container and sent to the laboratory as soon as possible. If a delay in transport or processing is anticipated, keep the specimen at 4^oC.

 

III. Procedure

 

A. Processing of Specimens:

Specimens should be set up as soon as possible or stored at 4ºC for up to 72 hours. All processed specimens should be stored at -70ºC after inoculation. Bloody fluids should be spun at 2000 rpm (700xg) for 10 minutes and the supernate used for inoculation.

Refer to Appendix II for Shell Vial inoculation and staining.

Refer to Appendix III for Tube Culture inoculation.

 

B. Direct Examination:

Not done.

 

C. Isolation and Identification:

Specimens	Method	Cell Linea	Incubation at 36oC	Stain used/Read
Amniotic fluid	Shell Vial	MRC-5 MRC-5 (if requested) MRC-5 (if requested) MRC-5 (if requested)	2 days 1 day 1 day 2 days	CMV-IE HSV1 HSV2 VZV
Shell Vial for CPE	E-Mix MRC-5 (summer**)	5 days 5 days	Read daily*** Read daily***
Pleural , Peritoneal , Pericardial, other fluids	Shell Vial	MRC-5 (if requested) MRC-5 (if requested) MRC-5 (if requested) MRC-5 (if requested)	2 days 1 day 1 day 2 days	CMV-IE HSV1 HSV2 VZV
Shell Vial for CPE	E-Mix MRC-5 (summer**)	5 days 5 days	Read daily*** Read daily***

^aMRC-5 = Human Fibroblast cells

** Summer= May to October

*** If CPE is detected, the technologist will pass the infected supernatant to a new shell vial and prepare a cell suspension of the infected culture so that multiple cell spots (on slide) can be stained with the enterovirus D3, Coxsackie B and ECHO stains to identify the virus.

 

D. Interpretation and Processing of Cultures:

a) For shell vial procedure:

i) For amniotic fluid (or other fetal specimens), always set up CMV shell vial. Fix and stain after 2 days (or next working day).

ii) For other fluids, set up CMV, HSV1, HSV2 and VZV if requested. Fix and stain as indicated.

See Appendix II for detailed shell vial procedure.

b) Shell Vial Cultures for CPE:

Shell Vials for CPE should be examined daily for Cytopathic effect (CPE). Any culture demonstrating 2+ or more CPE should be confirmed using appropriate monoclonal antibodies immunofluorescent staining (Refer to Appendices IV and V). If positive, record in freezer program and freeze the cells and supernate (Refer to Appendix X and XII).

c) Any culture demonstrating CPE for which a virus cannot be detected using monoclonal antibodies or other in-house methods and toxicity has been ruled out (see below) should be referred to the Public Health Laboratory (PHL) for electron microscopy and further work-up. Consult the charge/senior technologist or medical microbiologist.

d) Culture Toxicity: If toxicity is suspected in a tube culture (rounding of cells, sloughing of cells, granular cytoplasm of cells or unusual CPE), the cells should be scraped and appropriate monoclonal antibody staining performed. Negative stain results indicate the need for a passage. Scrape cells and add 0.2 ml of these scraped cells to a fresh tube containing 2 ml of media (1:10 dilution) and proceed again with tube culture method. (Appendix III). If toxicity or CPE persists, refer to the charge/senior technologist for review.

e) Contaminated Culture: If the tube culture is visibly contaminated and uninterpretable, replant the specimen.

Refer to Appendix II for Shell Vial staining interpretation.

Refer to Appendix III for Tube culture reading and interpretation.

 

IV. Reporting Results

Shell Vial: Negative Report: “Negative for ______virus.”

Positive Report*: “POSITIVE for _______virus.”

Tube Culture: Negative Report: “No virus isolated”

Positive Report*: “POSITIVE for _______virus.”

Toxicity Report: "Virology Tube Culture: Specimen toxic to cell culture.

Contaminated Report: "Virology Tube Culture: Specimen is heavily contaminated with bacteria and/or fungus. Unable to perform Virology Tube Culture.

 

V. Reference

1. Gleaves, Curt A. et al. Cumitech 15A “Lab Diagnosis of Viral Infections”.

American Society for Microbiology, August 1994.